Method for detecting urine metabolite based on deriving method

A detection method, urine technology, applied in the field of detection of urine metabolites, can solve problems such as undiscovered body metabolites, and achieve fast response, high reproducibility, and good stability

Inactive Publication Date: 2008-06-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The derivatization method of methyl chloroformate has been used in the determination of amino acids and organic acids in the past. In further searches, no relevant literature reports on the use of metabolites in the body have been found.

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  • Method for detecting urine metabolite based on deriving method
  • Method for detecting urine metabolite based on deriving method
  • Method for detecting urine metabolite based on deriving method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Rat urine samples stored at -80°C (24-hour urine samples from rats collected in metabolic cages) were taken, thawed and centrifuged to remove precipitates. Take 600 μL of the supernatant, add 400 μL of methanol, 100 μL of pyridine, and add 50 μL of methyl chloroformate, and perform derivatization reaction on a 40KHz ultrasonic instrument for 60 s. Take 300 μL chloroform to extract the derivatized mixture. And add 7mol / L 100μL sodium hydroxide solution, add 50μL methyl chloroformate to the above solution, mix, and perform secondary derivatization reaction on a 40KHz ultrasonic instrument for 60s; , centrifuged for 10min (3000 rev / min), discarded the water layer, and dried the remaining chloroform layer with a small amount of anhydrous sodium bisulfate to be determined by gas chromatography-mass spectrometry. The results are shown in Figure 1. From the analysis, 85 metabolites in urine were identified, including 20 kinds of amino acids, 32 kinds of organic acids, 12 kin...

Embodiment 2

[0032] Take urine samples from mice stored at -80°C (24-hour urine samples from mice collected in metabolic cages), thaw and centrifuge to remove the precipitate. Take 600 μL of the supernatant, add 400 μL of methanol, 100 μL of pyridine, and 50 μL of methyl chloroformate, and perform derivatization reaction on a 40KHz ultrasonic instrument for 60 s. Take 300 μL chloroform to extract the derivatized mixture. And add 7mol / L 100μL sodium hydroxide solution, add 50μL methyl chloroformate to the above solution, mix, and perform secondary derivatization reaction on a 40KHz ultrasonic instrument for 60s; after the reaction is completed, the reaction solution is vortexed for 30s, Centrifuge for 10 min (3000 rpm), discard the water layer, and dry the remaining chloroform layer with a small amount of anhydrous sodium bisulfate for determination by gas chromatography-mass spectrometry. The measurement results are shown in Figure 2. From the analysis, 45 metabolites in urine were ident...

Embodiment 3

[0034] Take guinea pig urine samples stored at -80°C (guinea pig urine samples collected in metabolic cages for 24 hours), thaw and centrifuge to remove the precipitate. Take 600 μL of the supernatant, add 400 μL of methanol, 100 μL of pyridine, and 50 μL of methyl chloroformate, and perform derivatization reaction on a 40KHz ultrasonic instrument for 60 s. Take 300 μL chloroform to extract the derivatized mixture. And add 7mol / L 100μL sodium hydroxide solution, add 50μL methyl chloroformate to the above solution, mix, and perform secondary derivatization reaction on a 40KHz ultrasonic instrument for 60s; , centrifuged for 10min (3000 rev / min), discarded the water layer, and dried the remaining chloroform layer with a small amount of anhydrous sodium bisulfate to be determined by gas chromatography-mass spectrometry. The measurement results are shown in Figure 3. 91 metabolites in urine were analyzed and identified, including 18 kinds of amino acids, 35 kinds of organic acid...

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Abstract

The invention relates to a measuring method of urine metabolic characteristic substance based on derivation method in the bioengineering technical field, including the following steps that: collected urine is stored after centrifugation; methanol, pyridine and methyl chloroformate are added in the urine and then derivation reaction of the mixture is carried out under the ultrasonic action of an ultrasonic instrument; chloroform is adopted to complete extraction of the derivation mixed solution and then sodium hydroxide solution is added in the mixed solution; methyl chloroformate is added in the obtained solution to be mixed with the solution once again; moreover, secondary derivation reaction is carried out under the ultrasonic action of the ultrasonic instrument; after the reaction, rotating recentrifuging of the reaction solution is carried out to eliminate the aqueous layer, and the remaining chloroform layer is dried by a little anhydrous sodium bisulphate to be used for gas phase chromatogram mass spectrum combination measuring; finally, the chloroform layer is used for gas phase chromatogram mass spectrum combination measuring. The invention has the characteristics of simple operation (capable of realizing reaction in water phase), rapid reaction (with ultrasonic reaction completed within 2 minutes), outstanding stability and high reproducibility, etc.

Description

technical field [0001] The invention relates to a method for detecting urine metabolites in the technical field of bioengineering, in particular to a method for detecting urine metabolites based on a derivation method. Background technique [0002] The determination of metabolites in urine is of great significance for understanding the normal physiological process, the mechanism of disease, the target of drug treatment, and the evaluation of drug efficacy. The measurement of metabolites has been used to understand disease since the middle of the last century. For example, a classic example is phenylketonuria. People detect whether the human body has a genetic defect by detecting phenylpyruvate in the urine. In recent years, metabolomics, which mainly focuses on the determination of metabolites in body fluids, has pushed the detection of metabolites to a new stage. People have tried to use various advanced instruments and methods to measure metabolites in body fluids, includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N1/28
Inventor 贾伟邱云平淡墨李后开
Owner SHANGHAI JIAO TONG UNIV
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